Abstract

Zika virus (ZIKV) has evolved into a global health threat due to its unexpected causal link to microcephaly. Phylogenetic analysis reveals that contemporary epidemic strains have accumulated multiple substitutions from their Asian ancestor. Here, we show that a single serine to asparagine substitution (S139N) in the viral polyprotein substantially increased ZIKV infectivity in both human and mouse neural progenitor cells (NPCs), led to more significant microcephaly in the mouse fetus, and higher mortality in neonatal mice. Evolutionary analysis indicates that the S139N substitution arose before the 2013 outbreak in French Polynesia and has been stably maintained during subsequent spread to the Americas. This functional adaption makes ZIKV more virulent to human NPCs, thus contributing to the increased incidence of microcephaly in recent ZIKV epidemics.

Zika virus (ZIKV) was first isolated from a sentinel monkey from the Zika forest, Uganda, in 1947 and until its recent emergence in the Americas, was known as an obscure mosquito-borne flavivirus (1). In decades following its discovery, sporadic human ZIKV infections with mild signs and symptoms were reported in a few countries in Africa and Southeast Asia. After several explosive outbreaks in Micronesia in 2007 and French Polynesia in 2013-2014, ZIKV rapidly swept through South and Central America in 2015. Early in 2016, the World Health Organization declared the current ZIKV epidemics a Public Health Emergency of International Concern owing to its unexpected causal link to congenital brain abnormalities, especially microcephaly during pregnancy (2, 3). Investigations with clinical materials and animal models have provided solid evidences that ZIKV directly targets neuronal progenitor cells (NPCs), leading to microcephaly as well as other severe pathological outcomes (4–9).

ZIKV contains a positive-sense 11 kb genome RNA that encodes three structural proteins (C, prM and E) and seven non-structural proteins. ZIKV strains can be divided into the African and Asian lineages (10), and the contemporary strains circulating in Pacific islands and the Americas are likely to have evolved from a common ancestral strain in Southeast Asia (11). However, there is serological evidence that ZIKV had been circulating in Southeast Asia for many years (10, 12). Why was microcephaly not recognized earlier? Besides the potential impact of herd immunity and the lack of diagnostics and surveillance in epidemic areas, one plausible hypothesis is that ZIKV has acquired some adaptive mutations to become more virulent to the human fetal brain. Some preliminary results from cell lines indicate strain-specific effects in ZIKV-infected cells (13, 14). Whether contemporary ZIKV strains are more likely to cause severe microcephaly in the fetus remains an open question.

Here, we first investigated the in vivo neurovirulence phenotypes of three contemporary ZIKV strains (MTQ/2015, VEN/2016, and SAM/2016) isolated in 2015-2016 (6, 15, 16) and compared with their Asian ancestral strain (CAM/2010) isolated in Cambodia in 2010 (17) in one-day-old neonatal mice, comparable to the third trimester of pregnancy in humans. Remarkably, upon intracerebral (IC) injection (18, 19), all three contemporary strains led to 100% mortality in neonatal mice with typical neurological manifestations, including inactivity, motor weakness and bilateral hind limb paralysis, while CAM/2010 killed only 16.7% animals (Fig. 1A). Nevertheless, the contemporary strain VEN/2016, showed similar growth and plaque characteristics as those of CAM/2010 in in vitro cell culture based studies (fig. S1). These results show that the contemporary strains of ZIKV are more neurovirulent in mice than their ancestral strain CAM/2010.

To determine whether the observed neurovirulence phenotype is directly associated with microcephaly, we further compared the contemporary and ancestral strains in an established mouse embryonic microcephaly model (4). Littermate brains were infected with the two ZIKV strains at embryonic day 13.5 (E13.5), which corresponds to infection at the second trimester of pregnancy in humans; a time point that is associated with the development of fetal brain abnormalities (20). Inspection of the mice at embryonic day 18.5 (E18.5) revealed that VEN/2016 infection resulted in significantly microcephalic brains (Fig. 1B), including the thinning of the cortex (Fig. 1C and fig. S2A). The microcephaly phenotype caused by VEN/2016 is comparable to our previous findings (4) using another contemporary ZIKV strain, the SAM/2016. By contrast, CAM/2010 caused less severe symptoms. Although both viruses predominantly targeted the NPCs as described previously (4), VEN/2016 showed significantly enhanced replication in the brain compared with CAM/2010 (fig. S2, B and C). This result was further verified in primary cultured mouse NPCs (mNPCs, fig. S2D). Specifically, VEN/2016 induced more apoptosis (cells positive for the activated form of Caspase 3) in different regions of the brain, as well as loss of more mature and immature neurons (neurons positive for NeuN and Tbr1, respectively), by comparison with to CAM/2010 (fig. S3). Furthermore, we found that VEN/2016 disturbed the proliferation and differentiation of NPCs more significantly than CAM/2010, as indicated by the substantial decrease in phosphorylated histone H3 (P-H3) positive cells (Fig. 1D and fig. S4A) and cell cycle exit index (Ki67-BrdU+/BrdU+ cells) (fig. S4B). Thus, the contemporary ZIKV strain is more virulent to mouse brain and causes a more significant microcephaly phenotype in embryonic brain than the ancestral strain.

We then sought to identify the genetic determinants for the observed virulence phenotype. Genome sequence alignment using the ancestral strain CAM/2010 as a reference strain revealed that contemporary ZIKV strains harbor a number of substitutions of amino acids throughout the genome (fig. S5). We mapped these amino acid substitutions on a Maximum Likelihood tree (fig. S6) and found a positive correlation between the evolution of ZIKV of the Asian lineage and sampling dates (Fig. 2A). Further coalescent analysis indicated that multiple critical residue substitutions have arose at different time points before the South American outbreak and have been maintained stably in the contemporary epidemic strains (Fig. 2B and fig. S7). To identify potential virulence determinants, seven mutant viruses (T106A, S109N, N130S, S139N, K709R, A982V, and N3144S) were constructed based on the infectious clone of CAM/2010 (WT) (fig. S8A) as described previously (17). All seven mutant viruses were successfully recovered in BHK-21 cells, and their replication as well as plaque phenotypes were found to be similar to the WT virus in vitro (fig. S8, B and C). Strikingly, of all the mutants, the S139N mutant virus exhibited the greatest neurovirulence in neonatal mice (Fig. 3A). More importantly, a single reverse substitution, N139S, of the parental virus VEN/2016 substantially decreased mortality caused by the virus in neonatal mice (Fig. 3B and fig. S9). We further characterized the infectivity of the S139N and WT viruses in human NPCs (hNPCs, fig. S10) derived from human embryonic stem cells (5), and the results confirmed that S139N showed enhanced replication in hNPCs (Fig. 3C), and caused more extensive cell death compared to the WT virus (Fig. 3D). Similarly, the S139N mutant showed enhanced viral replication in mNPCs compared with the WT virus (fig. S11A).

Fig. 2Phylogenetic and molecular clock analysis of ZIKV strains of the Asian lineage.

(A) The root-to-tip analysis using TempEst v1.5. The input was a Maximum Likelihood tree estimated using RAxML with 1000 bootstrap replicates (fig. S4). (B) The Bayesian phylogenetic tree estimated using BEAST v1.8.4. The positions of CAM/2010, VEN/2016, SAM/2016 and MTQ/2015 were indicated with black and red arrows. Conserved amino acid changes were inferred using the CAM/2010 strain as the parental strain. The green bars indicated the 95% highest probability density intervals of the age of the lineage. The details of the tree are shown in fig. S5.

Finally, we tested the impact of the S139N substitution in the embryonic microcephaly model. Remarkably, infection of the S139N mutant virus led to a more severe microcephalic phenotype (Fig. 4A) and thinner cortex (Fig. 4B and fig. S11B) than the WT virus. Specifically, the S139N mutant virus showed a more robust infection of the NPCs of the embryonic brains (fig. S11, C and D), accompanied by more cell death in the cortical plate (Fig. 4C). Furthermore, the S139N mutant disturbed the NPC proliferation and differentiation more significantly than the WT virus (Fig. 4D and fig. S12). As expected, the N130S mutant virus caused much less cell death than the S139N mutant in different regions of the brain, similar to the WT virus (fig. S13).

Bioinformatic analysis had identified a panel of substitutions that could have given rise to the different biological phenotypes of ZIKV (11, 21, 22). However, none of these predications have been validated experimentally. Here, our results from reverse genetics and mouse neurovirulence studies provide experimental evidence that a single S139N substitution significantly enhances ZIKV infectivity in both human and mouse NPCs and leads to more significant microcephaly in fetal mice. Coalescent analysis indicated that the ZIKV S139N substitution first emerged in May, 2013 (95% highest probability density intervals: November, 2012~October, 2013), a few months before the 2013 outbreak in French Polynesia (21, 23), and was then stably maintained in the epidemic strain during subsequent spread to the Americas (Fig. 2). The emergence of the S139N substitution correlates with the emergence of reports of microcephaly and other severe neurological abnormalities, including Guillain-Barre syndrome (24, 25). Our findings offer an explanation for the unexpected causal link of ZIKV to microcephaly, and will help understand how ZIKV evolved from an innocuous mosquito-borne virus into a congenital pathogen with global impact.

Structural modeling based on dengue virus (DENV), a closely related flavivirus member, indicates that Residue 139, refers to as Residue 17 of prM protein, is fully exposed on the surface of prM-E heterodimers or immature particles (fig. S14). The prM protein of flavivirus is required for viral maturation, egress and secretion, and the pr domain is thought to prevent premature fusion within the infected cells (26). There is a mixture of immature, partially mature, and mature particles in flavivirus-infected cells (27). A recent study showed that the first 40 amino acids of the pr domain are involved in the interactions within trimeric spikes in the immature virus particle and affect the dynamics of conformational changes (28). The S139N substitution might have some effects on the transition of ZIKV from the immature to the mature virion and the heterogeneity in maturity of progeny virions might thus affect viral fitness as well as neurovirulence. Our results also show that the ancestral Asian strain CAM/2010 can result in mild microcephaly phenotype in mouse fetus (Fig. 1), and the N139S revere mutant virus of contemporary ZIKV strain retains some neurovirulence to neonatal mice (Fig. 3B), thus further work will be required to identify additional viral genetic determinants and host factors that might affect ZIKV pathogenesis. In addition, enhancement of vector infectivity by specific amino acid substitutions has been reported in ZIKV and other mosquito-borne viruses (29, 30), the potential relationship between epidemic potential and disease severity is being under investigation.

Acknowledgments: We thank Drs. Andrew Davidson (University of Bristol), Ai-Hua Zheng (CAS), and Bo Zhang (CAS) for helpful discussion and critical reagents. This work was supported by the National Natural Science Foundation (NSFC) of China (No.31770190, No.31730108, No.81661148054, and No.81661130162), the National Key Research and Development Project of China (No.2016YFD0500304), the National Science and Technology Major Project of China (No.2017ZX09101005 and No.2017ZX10304402), Chinese Academy of Sciences (QYZDJ-SSW-SMC007), Shanghai Brain-Intelligence Project from STCSM (16JC1420500), and Beijing Bain Project (Z161100002616004). C.-F.Q. was supported by the Excellent Young Scientist (No.81522025), Innovative Research Group (No.81621005) from NSFC, and the Newton Advanced Fellowship from the UK Academy of Medical Sciences. W.S. was supported by the Taishan Scholars program of Shandong province (ts201511056). All data to understand and assess the conclusions of this research are available in the main paper and supplementary materials.